Substantial evidence has demonstrated a role for the Notch gene family in multiple human cancers, including neoplasms of the lymphoid system, pancreas, breast and CMS, among others. However, the pathophysiological mechanism of Notch function remains poorly understood. Deregulation of the Notch signal transduction pathway can drive the neoplastic conversion of cells, playing an important role in both the initiation and maintenance of the transformed state. This transforming activity is an intrinsic property of Notch, which mediates its effects through a transcriptional cascade. The underlying hypothesis of this proposal is that Notch is a scaffold protein that mediates its function through the assembly of a transcriptional regulatory complex(s), which governs changes in gene transcription. Our goal is to characterize the molecular mechanisms regulating the assembly and stability of Notch complexes and Identifying mechanisms that mediate and/or regulate Notch function to better understand Notch signaling in normal and pathological conditions. Our preliminary data demonstrate that assembly and stability of Notch activation complex is regulated by Notch multimerization, phosphorylation and binding to other proteins. These mechanisms may regulate Notch activity through timing of the complex assembly, stabilization of the protein and/or differential activation of Notch targets. Specific aims for this proposal include; i) characterization of the critical parameters that regulate Notch multimerization and transcriptional complex assembly, ii) characterization of the role of a novel Notch-binding protein termed NACK (Notch Activation Complex Kinase) in Notch activity, and iii) characterization of posttranslational modifications of Notch that play a role in its transcriptional activity regulatio. The long-range goal for these studies is to obtain a comprehensive understanding of how Notch activity transforms cells in order to contribute to the rational design of cancer therapeutics. PUBLIC HEALTH RELEVANCE: Notch has been implicated in multiple human cancers, including neoplasms of the lymphoid system, pancreas, breast and CMS, among others. However, despite the involvement of Notch in the initiation and maintenance of tumors the molecular mechanisms of Notch activity are still poorly understood. The goal of this research proposal is to unveil these mechanisms and determine how Notch activity transforms cells in order to contribute to the rational design of cancer therapeutics.